Reerences aDd Notes 1. K. v. Frisch, Naturwissenschaften 35, 12 (1948); ibid. 38,105 (1951). 2. , The Dance Language and Orientation of Bees (Harvard Univ. Press, Cambridge, Mass. 1967). 3. , Osterr. Zool. Z. 1, 1 (1946). 4. E. 0. Wilson, The Insect Societies (Harvard Univ. Press, Cambridge, Mass., 1971), p. 266. 5. M. Lindauer, Communication among Social Bees (Harvard Univ. Press, Cambridge, Mass., 1961), pp. 59-86. We refer to Apis dorsata and A. florea rather than to A. indica which, though vaguely tropical, lives in cavities and is so similar toA. mell#era as frequently to be considered a subspecies. 6. M. Lindauer, Z. Vergl. Physiol. 37, 263 (1955). 7. K. v. Frisch, Experientia 5, 142 (1949). 8. M. L. Brines, thesis, Rockefeller University (1978). The light source was either a d-c. quartzhalogen or xenon-arc, with quartz optics. A quartz diffuser was used to make the radiance distribution quite uniform. Light was linearly polarized by a Polaroid HNP'B filter. Spectral distribution was controlled by band-pass filters, which included Wratten 2a, 15, IBA, and 45 as weR as Hoya 330, 370, and 390. Data for Fig. I was derived by projecting polarization patterns through an iris onto a UV-transparent, diffusing screen. Transmitted light retained its polarization. Dancing bees viewed the stimuli through a No. 10 open nylon mesh. A variety of controls established that no other cues were being utilized and that no unknown physical bias influenced the choice of dance direction. 9. Bees try to use any stimulus directly above them as if it is a part of the sky. A zenith sun has no azimuth, while a zenith sky pattern has only two possible interpretations. This exphins the conflict between Frisch's report (2, p. 402) that bees

10. 11.

12.

13.

14.

15.

16.

treat small sources of white, polarized light as the sun, and the experiments of W. Edrich and 0. von Helversen [J. Comp. Physiol. 109, 309 (1976)], for which bees used white, polarized zenith stimuli as part of the sky. The difference between Frisch's finding (1) that bees need at least 100 to IS' of blue sky in order to orient to polarized light, and those of Edrich and Helversen in which bees oriented weli to far smaller spots, is also explained. The natural sky used by Frisch made the data fall behind the 150 boundary of sky and sun in Fig. 1, while Edrich and Helversen's fall to the right of the 20 to 30 percent UV boundary. Bidirectional dances reported by others (1, 15) probably result from using stimuli near the boundary of sun and sky. Coloropponent interneurons with these characteristics have been found in bees by J. Kien and R. Menzel [J. Comp. Physiol. 133, 35 (1977)], and could be the innate releasing mechanism. P. Hess, Beitr. Geophy. 55, 204 (1939). Hess' data were reported in energy units that we have corrected to relative photon flux with respect to the spectral sensitivity of bees. M. L. Brines and J. L. Gould, J. Comp. Physiol., in preparation. J. Strutt (Lord Rayleigh), Phil. Mag. 41, 107 (1871); S. Chandrasekhar, Radiative Transfer (Clarendon, Oxford, 1950). Bees also appear to use a fourth rtle. When shown an E-vector orientation that does not exist at the elevation chosen (Fig. 2), they stili perform consistently oriented dances. We do not yet know how this rule works since the dance orientation does not seem to be predicted by any geometrical theory (for example, K. Kirschfeld and M. Lindauer, Z. Naturforsch. 30c, 88 (1975)] of polarization orientation in bees. Indeed, this ability on the part of bees suggests that they may not use the Rayleigh scattering relationships at all (8, 15). R. Boch, Z. Vergi. Physiol. 40, 289 (1957). S. Rossel, R. Wehner, M. Lindauer, J. Comp. Physiol. 125, (1978). These workers found that the dances were generally biomodal, but one direction was selected by the bees more frequently than the other. Our results show bimodality only rarely. The difference is that Rossel et al. probably used sources that fell close to the boundaries between sun and sky, while our stimuli were far from any boundary. We thank R. Alexander, R. Dahl, K. Schenk, D. Thompson, E. Tyner, and H. Wildman for technical assistance and C. G. Gould, R. M. Shapley, W. G. Quinn, and especially D. R. Griffin, for valuable discussions. Supported in part by NSF grnt BNS 76-01653 to J.L.G.

20 November 1978; revised

Ethanol Embryotoxicity: Direct Effects on Mammalian Embryos in vitro Abstract. Exposure to ethanol retards growth and differentiation in cultured rat embryos during organogenesis. The development of untreated embryos is indistinguishable from growth in utero. These data suggest that the hypoplastic-features of children born to chojcall alcoholic mothers are due, at least in part, to a direct causes reduced embryonic cellular proliferation early in action of ethano gestation.

Excessive use of alcoholic beverages results in a variety of medical, psychological, and sociological disruptions that identify alcoholism as one of modern society's major problems. Since a characteristic pattern of congenital malformations associated with the offspring of alcoholic mothers was described (1), attention has been focused on the toxic effects of alcohol consumption in pregnancy (2). This fetal alcohol syndrome (FAS) has now been observed in more than 200 infants, and the relationship to chronic alcoholism is well established SCIENCE, VOL. 206, 2 NOVEMBER 1979

(3). However, the means by which FAS

is produced are uncertain at present. It is not known if the developmental anomalies are the result of a direct action of ethanol or its metabolites on embryonic tissue, or if they are the product of altered matemal function, or a combination of such factors. In addition, it is not known if there is a sensitive period of gestation during which alcohol may exert teratogenic effects or if prolonged heavy drinking before pregnancy is a prerequisite for the complete FAS. We are currently evaluating the em-

bryotoxic potential of environmental agents in cultured rat embryos during a major portion of the organogenesis peric 1. The culture system supports embryonic growth and differentiation indistinguishable from that in utero. Orf g -iogenesis is thought to be the interval of greatest embryonic sensitivity to environmental factors, and congenital malformations are most likely to be the result of teratogenic insult over this period. Our studies have shown that in embryos cultured in the presence of ethanol, both differentiation and growth were retarded as a function of dosage, but no gross alterations in morphogenesis were induced. To our knowledge, this is the first un-

equivocal demonstration of a direct action of ethanol on the developing mammalian embryo, without the confounding factors of altered maternal function, nutrition, or metabolism. Our experiments were designed to investigate the development of embryos continuously exposed, during organogenesis, to ethanol at concentrations of 150 or 300 mg of ethanol per 100 ml of culture medium (4). Conceptuses were explanted from outbred rats (Charles River) during the afternoon of the tenth *day of pregnancy (embryonic age, 91/2 days) (5). All operations were carried out aseptically, and no antibiotics were used throughout the study. Embryos within the yolk sac and amnion were dissected free of maternal decidua and Reichert's membrane, the ectoplacental cone being left intact. Two conceptuses were cultured in 4 ml of medium (6) contained in 30-ml serum bottles. During culture, bottles were kept in gentle motion by use of a roiler apparatus (6), and the temperature was maintained at 37°C for the 48hour culture period. The oxygen concentration in the gas phase of the bottles was increased from an initial 5 percent 02 to 20 percent 02 at 17 hours, and 40 percent 02 at 26 hours (5 percent CO2 at all times, the balance N2). At least two conceptuses from each rat were randomly assigned to 300 mg of alcohol per 100 ml, 150 mg of alcohol per 100 ml, and control bottles. Alcohol was added to the medium at the beginning of the culture from a stock solution of ethanol which was at a concentration such that the osmolarity of the serum (305 mosmole/liter) was maintained (7). Control bottles received the same volumes of Hanks basic buffered salt solution isosmolar to the senm. At the end of the culture, embryos and their associated membranes were examined, measured, photographed, and frozen for subsequent biochemical analysis. To estimate differentiation and abnormal organogenesis, we have devised a com-

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necessary or even desirable for any of the vast number of social and nonsocial animals that perform the same feats of navigation, but lack a symbolic language (16). MICHAEL L. BRINES Rockefeller University, New York 10021 JAMES L. GOULD Department ofBiology, Princeton University, Princeton, New Jersey 08450

Table 1. Effect of ethanol on the in vitro growth of 9'/2-day rat conceptuses, given as means ± standard errors. Growth after 48 hours of culture

Embryo Crown-rumplength(mm) Head length (mm) Number of somites Total DNA (g) Total protein (,g) Yolk sac Diameter (mm) Total DNA (,g)

Control culture (N = 18) 4.54

t

2.26 29.2 33.4 333.3

± ± ± ±

0.08 0.04 0.23 1.68 13.5

Ethanol per 100 ml of culture medium 150 mg 300 mg (N = 11) (N = 13)

4.29 2.15 28.6 31.9 295.4

t

0.12

± 0.05 ± 0.37 ± 1.81 ± 16.7

3.78 1.84 26.6 22.7 223.4

t

± ± ± ±

0.l0*t 0.07*t 0.51*t 2.13*t 17.1*t

Totalprotein(,ug)

5.07 ± 0.08 8.58 ± 0.28 169.8 ± 5.88

5.05 ± 0.16 9.04 ± 0.71 166.0 ± 14.4

4.75 ± 0.14 8.61 ± 0.55 151.2 ± 9.37

Placenta Total DNA (,ug) Total protein (,ug)

4.02 ± 0.36 60.3 ± 7.70

4.68 ± 0.76 96.8 ± 19.2

3.71 ± 0.50 59.8 ± 14.6

*Significantly different from control values and from 150 mg/100 ml values (pairwise Mann-Whitney U test, P < .01). tSignificant dose response (Jonckheere's test, P < .01).

prehensive morphological scoring

sys-

tem to grade the development of the yolk

placenta, and embryonic organ primordia according to observable morphologic features (8). This system makes it possible to determine embryonic development with an accuracy equivalent to + 2 hours of gestation. Total protein and DNA contents (9) were measured after the tissues were homogenized by sonication. Over the 48-hour period from embryonic age 9'/2 days to ll'/2 days, the rat embryo develops from the, early neurula stage with 0 to 3 somites to the tail bud stage with 28 to 30 somites (Fig. 1). This period is equivalent to approximately 10 days of human embryonic development, sac,

from 20 days to 30 days of gestation. Tissues become extensively segregated into the primordia of the neural, sensory, cardiac, circulatory, and hepatic organs. Within this culture system, the growth of embryos in vitro was indistinguishable from growth during the equivalent period in vivo (10). Growth of the ectoplacental cone was severely reduced in vitro; nevertheless, a vigorous, functional, chorioallantoic placental circulation was established in cultured conceptuses. Embryos cultured in the presence of ethanol showed a marked reduction in growth. The embryonic growth measures of length from crown to rump, total DNA, and total protein contents were significantly reduced in the 300-mg al-

Fig. 1. Rat embryos at age 9'/2 days (A) and after 48 hours of culture (B). (A) and (B) are at the same magnification and show the extensive growth over the culture period. (C) A 9'/2-day embryo at x2.5 greater magnification to illustrate the relative lack of differentiated tissue at this stage. 574

cohol group, with a significant doseresponse trend in all cases (Table 1). Growth measures for the yolk sac and placenta were not affected by the presence of alcohol. Not only was embryonic growth reduced, but differentiation was also retarded as a function of dosage. The morphological scores were 41.6 + 0.4 for control embryos, 40.7 + 0.3 for the 150-mg group, and 38.4 ± 0.8 (significantly different from control, P < .02) for the 300-mg group. Retarded development was also indicated by the dosedependent reduction of the mean number of somites developed. Observations detected microcephalic growth of treated embryos, illustrated by reduced head lengths (Table 1). No gross structural defects were observed in either treated or control embryos. Comparing the growth and development measures of embryos in the 300-mg group with equivalent measures for control cultures, we estimated that treated embryos were retarded by 5 to 7 hours of gestation. This value is consistent, whether based on morphological, mensural, or biochemical variables. From total DNA concentrations, we have calculated the cell contents and kinetics of cultured embryos (11). Embryos treated with 300 mg of alcohol per 100 ml have a deficiency of about 8.9 x 105 cells, compared with control embryos. This result is consistent with the calculated 5- to 7hour retardation, which represents approximately two-thirds of the cell cycle time at this stage of gestation (11). The ratios of total DNA to total protein contents were not significantly affected by ethanol treatment, which suggests that cell size was not altered. Investigations in our laboratories have demonstrated that the culture of rat embryos, according to this method, can be a sensitive system to detect developmental malformations. For example, dimethadione, the major metabolite of the anticonvulsant trimethadione, a known rodent and human teratogen (12), induced abnormalities of neural tube closure, cardiogenesis, mesoderm segmentation, cephalocaudal flexion, and brain stem development at concentrations of 2.5 to 10 mM (13). (By comparison, 300 mg of ethanol in 100 ml is a 33 mM solution.) In contrast, no gross defects were observed in embryos cultured in the presence of ethanol. However, developmental retardation during gestation, as seen in this study, is consistent with the major manifestations of FAS. The most frequent phenotypic features of FAS (prenatal and postnatal growth deficiencies, microcephaly, short palpebral fissures, mandibular and midfacial growth reducSCIENCE, VOL. 206

43 (1975)], the embryo of a chronically alcoholic mother is likely to be exposed to such concentrations of alcohol for extended periods. day on which sperm was detected in vaginal s. The smears was designated day 1. Embryonic ages were calculated under the assumption that fertiloccurred at the midpoint of the dark izaton cce(midnight). New,48, P. T.219Coppola, D. L. Cockroft,J. 6. Reprod. D. A. T. Fert. (1976). The medium was a

tions) are hypoplastic (3). Our observations suggest that these structural deficiencies may be the result of reduced cellular proliferation in the organogenesis phase, due to a direct action of ethanol (14). Clinical correlation of head size at birth with subsequent brain function has

homologous serum, immediately centrifuged

inactivated by P. T. Coppola, Teratology suggested that microcephaly is strongly 7. and D. L. Cockroft andheat. related to mental retardation (15). Since 16, 141 (1977). 8. N. A. Brown, E. H. Goulding, S. Fabro, in we observed microcephalic growth in 0. H. Lowry, N. J. Rosebrough, A. L. Farr, R. this study, the mental retardation seen in 9. preparation. Chem. 193,Anal. 265 (1951); U. bothand fully nd partially partiallexpressedJ. Randall, FSKsnA >lheegerAnS both fully Karsten andJ.BS) A.Biol. Biochem. expressed FAS Wollenberger, 464 (1977). (16) may be the result of a direct inhibi- 10. 77. D. A. T. New, P. T. Coppola, D. L. Cockroft,J. tion by ethanol of neural growth early m Embryol. Exp. Morphol. 36, 133 (1976). 11. Cell numbers were calculated by assuming 12 pg gestation. of DNA per embryonic cel [E. Kohler, H.-J. Our demonstration of ethanol-induced'. Merker, W. Ehmke, R. Wojnorowicz, NaunynSchmiedeberg's Arch.cell-cycle Pharmacol. 272, esti169 developmental retardation suggests that times were (1972)]. Approximate mated from mean cell numbers of embryos. FAS may not be the result of maternaly taken times,increased over days 9'/2 to 12'/2 of gestation. or altered materproduced metabolites days produced ~~~~~~~~~~~cycle from 6.2 hours at 9'/2 Cellto 10.6 hours at 12 days, an increase compatible nal function. Whether the embryotoxic . with characterization of embryonic growth the agent is ethanol itself or some other speas Gompertzian [A. K. Laird, Growth 30, 263 cies produced by embryonic metabolism .12. 1.(1966)]. German, A. Kowal, K. Ehlers, Teratology 3, of ethanol is not yet clear. Current evi349 (1970); N. A. Brown, G. Shull, S. Fabro, Toxicol. Appl. Pharmacol., in press. dence, however, shows that embryos at this stage of gestation do not possess any ethanol-oxidizing or alcohol dehydrogenase activities (17). Although our reseen*

-

13. N. A. Brown and S. Fabro, Proceedings of the 26th Annual Meeting of the Society for Gynecological Investigation 132, A219 (1979). 14. Less common features of FAS are a mnge of minor and major malformations such as ventricular and atrial septal defects, cleft lip, cleft palate, and microphthalmia (3). These defects may also be the result of reduced growth of specific tissues during organogenesis, which is manifested later in gestation as dysmorphogenesis. Reduced ccllular proliferation has been proposed as the mechanism of action of several teratogenic agents, particularly those which cause maltormations such as cleft palate [W. J. Scott, in Handbook ofTeratology, J. G. Wilson and F. C. Fraser, Eds. (Plenum, New York, 1977), vol. 2, pp. 81-98]. 15. L. Crome, in The Brain in Unclassified Mental Retardation, J. B. Cavanaugh, Ed. (Churchill Livingstone, London, 1972), p. 284; A. Milunsky, The Prevention of Genetic Disease and Mental Retardation (Saunders, Philadelphia, 1975), pp. 19-50. 16. A. P. Streissguth, C. S. Herman, D. W. Smith, J. Pediatr. 92, 363 (1978). 17. P. H. Pikkarainen and N. C. R. Raiha, Pediatr. Res. 1, 165 (1967); N. C. R. Raiha, M. Koskinen, P. Pikkarainen, Biochem. J. 103, 623 (1967); A. K. Rawat, Ann. N. Y. Acad. Sci. 273, 175 (1976). 18. We thank the Audiovisual Services Department of George Washington University Medical Center for Fig. 1. * Send reprint requests to N.A.B. at George Washington University Medical Center. 9 March 1979; revised 11 June 1979

sults demonstrate that continuous ex- Water, Protein Folding, and the Genetic Code

posure to high levels of ethanol exerts a direct toxic action on the developing embyro, the effects of short-term ethanol exposure have yet to be determined. NIGEL A. BROWN* Laboratory of Environmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, and, Department of Pharmacology, George Washington University Medical Center, Washington, D.C. 20037 EUGENIA H. GOULDING

Abstract. The absolute affinities of amino acid side chains for solvent water closely match their relative distributions between the surface and the interior of native proteins and are associated with a remarkable bias in the genetic code.

Many processes of biological "recognition" require the stripping away (at, least in part) of solvent water from interacting groups. The mutual affinities therefore reflect in part the ease with which they can be removed from solvent water, in addition to any specific forces of attraction or repulsion that may be present. We now report the free-energy changes associated with the removal of Laboratory of Environmental side chains of common amino acids from Toxicology, National Institute solvent water. These changes resemble of Environmental Health Sciences SERGIO FABRO the relative distributions of the amino Laboratory ofEnvironmental Toxicology, National Institute of Environmental Health Sciences, and Nitrogen. Departments ofPharmacology and Obstetrics and Gynecology, George Washington University Medical Center Reerences and Notes 1. Bureau of Alcohol, Tobacco and Fireanns, The Fetal Alcohol Syndrome: Public Awareness Campaign (Government Printing Office, Wash-

ington, D.C., 1979). 2. P. Lemoine, H. Haronsseau, J.-P. Borteyru, J.C. Menuct, Quest Med. 25, 476 (1968); K. L. Jones, D. W. Smith, C. N. Ulleland, A. P. Streissguth, Lancet 1973I, 1267 (1973). 3. S. Clarren and D. W. Smith, N. Engl. J. Med. 298, 1063 (1978). 4. Alcohol concentrations were chosen to mirror those blood alcohol concentrations observed following heavy alcohol consumption in humans [F. G. Hoffman, A Handbook on Drug and Alcohol Abuse (Oxford Univ. Press, New York, 1975), p. 1021. Since ethanol passes freely into the embryonic compartment [Y. A. Kesaniemi and H. W. Sippel, Acta Pharmacol. Toxicol. 37, SCIENCE, VOL. 206, 2 NOVEMBER 1979

Pots containing radioactive solute

RHvapor _H Keq RH aqueous

acids between the surfaces and the interiors of native globular proteins, and are associated with a sharp bias in the genetic code. The affinity of a compound for watery surroundings can be expressed quantitatively in terms of its free energy of transfer from the dilute vapor phase, in which intermolecular forces are virtually absent, to an aqueous solution so dilute that solute-solute interactions can be neglected. Results obtained for many compounds suggest that this measure of the

Spray trap

Traps for collecting radioactive solute

Fig. 1. (Left) Water-vapor distribution coefficient; (above) apparatus for determining the partial pressure of polar solutes (2)

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575